The overall goal of this research is to develop a mechanistic understanding of the vacuolar biogenesis pathways in the yeast Saccharomyces cerevisiae. Genetic analysis has led to the identification of a very large number of components required along the vacuolar assembly pathway in yeast. A complex of proteins called GARP has been identified that functions in retrograde membrane transport to the late-Golgi compartment, and this complex will be investigated further both genetically and biochemically. GARP is one of the simplest and best characterized tethering complexes, and a complete molecular, biochemical and structural analysis of GARP should provide considerable insight into the role of tethering complexes in vesicle fusion. A genome-wide screen for new VPS genes has identified over 20 new genes/proteins involved in vacuolar biogenesis, and a number of these genes will be investigated further. Eight of these new Vps proteins are involved in transport steps between the Golgi and endosome, and will be investigated by genetic and biochemical approaches. Four of these new Vps proteins are involved in functions related to the prevacuole, and the network of interactions between these Class E Vps proteins will be investigated to better understand the relationship between multivesicular body formation and vesicle transport from the lateendosome/ prevacuole. This work also uncovered two new non-SNARE-like small Vps proteins predicted to span the bilayer four times, and these proteins will be investigated for a role in recruiting hydrophilic Vps protein complexes to the membranes where they function. Studies of membrane traffic in yeast have proven tremendously useful to a broader understanding of membrane transport in all eukaryotic cells because of the remarkable similarity in mechanisms and proteins that regulate these processes from yeast to humans. These basic mechanistic studies in yeast are providing important insights into our understanding of many lysosomal storage diseases in humans.